This invention relates to a method useful for removing citrate, aluminum and other multivalent ions from biologically active proteins. The method is particularly useful for removing aluminum and citrate ions from solutions containing albumin.
Biologically active proteins are frequently administered to humans as therapeutic agents. It is important that such proteins be free from contaminants that may cause adverse effects. It is known, for example, that purified human serum albumin (albumin) used widely in solutions intended for intravenous administration and as a plasma volume expander, may contain levels of aluminum that are unacceptable for use in humans.
The presence of aluminum in humans has been linked to senile dementia of the Alzheimer type and to neurofibrillary degeneration. Aluminum administered intravenously can accumulate in tissues and organs, such as the brain, and poses a particular threat to patients with impaired renal function who are unable to adequately eliminate the aluminum from the body. In such patients, aluminum contamination of dialysis solutions has been linked to osteomalacia, microcytic anemia and dialysis encephalopathy. As a consequence, albumin sold in Europe for intravenous administration is required to have a level of aluminum less than or equal to 200 ppb in solutions having 5, 20 or 25% protein concentration, a level that should be maintained throughout the dating period of the albumin product. The adverse effects of other metals, such as iron, lead, mercury, chromium, copper and nickel, have also been documented.
It has been shown that albumin acquires aluminum from a number of sources, including the diatomaceous earth used during albumin processing, glass containers, clay-filled elastomeric enclosures, and depth filters containing diatomaceous earth. (Quagliaro, D. A. et al., Aluminum in Albumin for Injection, Journal of Parenteral Science and Technology, 42(6), 187-190 (1988)).
Some solutions show an increase in the level of aluminum during storage, attributed to the extraction of aluminum from glass containers. Glass containers appear to be a significant source of aluminum contamination, as many such containers are composed of 1 to 5% aluminum. Factors that contribute to the level of aluminum in a protein solution are the storage conditions of the solution in glass containers and the nature of solutes present in the protein solution. For example, carboxylic acids which have an alpha hydroxy group, such as citrate anions, are good chelators of metal ions and are well known for their solubilizing effect on aluminum-containing substances.
Citrate ions are introduced into plasma-derived proteins during the normal plasmapheresis procedure, which involves the collection of plasma in the anticoagulant sodium citrate. For example, solubilized Cohn Fraction V powder, the starting material in the preparation of albumin solutions by the acetone process, has a citrate content of about 7.6 mM to about 9.7 mM (about 2235 ppm to about 2853 ppm). Thus, in addition to ensuring a low level of aluminum in albumin and other protein solutions to be administered to humans, it is also important to ensure a low level of citrate to avoid possible leaching of aluminum from glass containers by citrate ions during storage.
Multivalent aluminum ions, as with other multivalent ions, bind to proteins, and attempts to remove these ions with chelating agents such as EDTA have been largely unsuccessful. Ultrafiltration dialysis techniques have been used to remove multivalent ions from proteins such as albumin. These protocols rely on the displacement of the multivalent ions bound to the protein by monovalent ions during dialysis. For example, U.S. Pat. No. Re. 36,259 describes the use of a 3% aqueous salt solution, such as sodium chloride or sodium acetate, in a diafiltration system to displace aluminum ions from albumin. Similarly, U.S. Pat. No. 5,229,498 describes the displacement and removal of multivalent ions from proteins by diafiltration against an aqueous solution containing monovalent alkali metal ions or ammonium ions in a concentration from about 0.15 M up to saturation.
In the above cases, however, the diafiltered protein contains bound monovalent ions. As a consequence, it is necessary to subject the protein to an additional round of diafiltration, usually against deionized water, to remove the monovalent ions. A method in which both citrate ions and aluminum ions, as well as other multivalent ions, are removed from proteins without the necessity for a second procedure to remove the monovalent ions would thus be preferred.
The present invention is directed to a process for removing citrate, aluminum, and other multivalent ions and contaminants from proteins by adjusting the pH of a solution containing the protein to a pH from about 7 to about 10, and diafiltering the pH-adjusted solution against pure water.